To establish the mouse IgGFc Receptor(moFcγRII)stable expression cell line.The coding sequence of FcγRII was amplified and cloned into the mammalian expression vector pcDNA3.0 under control of the CMV promoter.The COS-7 cells were tranfected with the recombinant plasmids,and the FcγRII stable expression COS-7 cells were selected by the antibiotics of G418,and subcloned by well-known method of limiting dilution.Rosetting test of the binding of mouse IgG-sensitized chicken erythrocytes show that the percent of positive cells were more than 90%.The establishment of the mouse FcγRII stable expression cell line laid the necessary foundation for the identification of the linear epitope of IgG binding on mouse FcγRII,and further investing the immunologic mechanism of Fc receptor of IgG.

Neutrophils and macrophages in cattle express a novel class of immunoglobulin Fc receptor, specific for bovine IgG2, termed boFc gamma 2R. In cows, the ability of neutrophils to kill immunoglobulin-opsonized microorganisms appears to depend largely on this subclass. Although related to other mammalian Fc gamma Rs, boFc gamma 2R belongs to a novel gene family that includes the human killer Ig-like receptor and Fc alpha RI (CD89) proteins. In this study, we describe the presence and characterization of this novel class of Fc gamma R in sheep. The comparative analysis of this novel Fc gamma R has allowed us to begin an exploration of some immunological characteristic of ruminants.

The extracellular domain of the boFcgamma2R gene was constructed and cloned into the Escherichia coli expression vector pET-28a. The recombinant protein was expressed at high level in E. coli BL21(DE3) and existed mainly as inclusion bodies. The inclusion bodies were solubilized in 6 M guanidine hydrochloride and refolded by rapid dilution. After renaturation, the purity of the recovered recombinant protein was up to 95%. ELISA assay showed that the renatured recombinant protein could inhibit bovine IgG2 binding to expressed boFcgamma2R on the COS-7 cell surface with an IC50 value of 0.68 microM. The overall yield of the active rsbo2R was up to 20 mg/l of culture. Crystals of the rsbo2R were grown at 293 K by the hanging-drop vapour diffusion method showed weak diffraction.

In this experiment we firstly improved the IBDV purification procedure with the help of IBDV rapid detection strip. Then we optimized the key dements:inoculation dose and the infection duration in immunohistochemistry.Using the biotin la- beled anti-IBDV mAb F22EA6 and the HRP labeled streptavidin to stain the IBDV infection positive cell dose not produce nonspecific result in suitable condition.The IBDV infection positive cell can be enumerated to titer the virus relatively and to some extent it can take place the method of TCID50.It also could be used to detect the IBDV infection positive CEF.

Based on the reported nucleotide sequence of IBDV in GenBank,a pair of primers that can amplify the cDNA of IBDV VP2 gene hypervariable region were designed.By RT-PCR,the VP2 gene was isolated from Xinxiang chicken(XX strain),and then cloned to pGEM-T easy vector for sequence analysis.The results revealed that XX strain was most closely related to the vvIBDV UK661 and OKYM,showing the nucleotide similarity of 98.73% and 99.15%,separately and the amino acid idendity of 100%,but different from other serotype I strains.

目的:构建牛Fcγ2R胞外区基因的原核表达载体,并在E.coli BL21(DE3)中表达.方法:提取健康成年牛的白细胞总RNA,经RT-PCR扩增牛Fcγ2R胞外区基因片段,并将其克隆到载体pGEM-T Easy,经限制性内切酶EcoR I和 Not I双酶切鉴定及序列测定后,再将其亚克隆入原核表达载体pET28a中,构建重组质粒pET-2R,转化E.coli BL21(DE3)经IPTG诱导表达组氨酸融合蛋白.结果:获得682 bp的编码牛Fcγ2R胞外区基因片段.以构建的重组质粒pET-2R转化E.coli BL21(DE3)后,经IPTG诱导,表达出相对分子质量(Mr)约为30 000的重组蛋白.SDS-PAGE分析显示,表达的蛋白主要以不溶性包涵体的形式存在于E.coli BL21(DE3)的胞质中,Western blot检测表明该蛋白能和牛IgG2结合.结论:成功地构建了原核表达载体pET-2R,并表达出重组蛋白.为研究牛IgG和受体的相互作用机制及其介导的免疫反应打下了基础.

A translucent and superhydrophobic surface was prepared with a sol-gel method on a glass substrate. The coating solution was obtained by hydrolysis of a chelate compound of aluminum isopropoxide and ethyl acetoacetate. Then, the films were prepared by dip-coating the glass substrates and subsequent surface modification with stearic acid. Field-emission scanning electron microscopy (FE-SEM), atomic force microscopy (AFM), Fourier transform infrared (FT-IR) spectroscopy, UV-Vis spectroscopy, and contact angle (CA) measurements were used for the surface characterization. It was demonstrated that the coatings comprised a roughness structure on two levels with the root mean square roughnesses (R-rms) of ca. 30 and 200 nm, resulting in water static contact angle up to 168 degrees and a small sliding angle of only 3 degrees. The average visible light transmittance of the coatings was 62%.

目的:建立稳定表达人FcγRⅡ(human Fc gamma receptor Ⅱ ,huFcγRⅡ)的真核细胞系,为后续研究奠定基础.方法:采用RT-PCR方法从人外周血白细胞合成FcγRⅡcDNA,构建huFcγRⅡ表达质粒pcDNA3-huFcγRⅡ,经酶切和PCR鉴定后,将pcDNA3-huFcγRⅡ质粒通过脂质体转染法转染COS7细胞,并经G418筛选获得稳定表达huFcγRⅡ的细胞克隆.采用免疫荧光法和玫瑰花环试验检测huFcγRⅡ的表达.结果:构建的pcDNA3-huFcγRⅡ真核表达质粒经酶切鉴定与预期一致,稳定转入COS7细胞后,经免疫荧光染色,约90%的转染细胞可见荧光,而未转染的COS7对照细胞未见荧光.结论:成功构建pcDNA3-huFcγRⅡ真核表达质粒,建立了稳定表达huFcγRⅡ的细胞系,为进一步研究奠定了基础.

Objective To study the effect of soluble, refolded, recombinant extracellular domain of the human Fc gamma receptor Ⅱ a (huFcγRⅡa) on the binding of human IgG to cells. Methods Extra-cellular domain of the huFcγRⅡ a gene was amplified from recombinant plasmid pe3huR Ⅱ by PCR and then cloned into pET-28a vector. The recombinant plasmid pETshuR Ⅱ was transformed into E. coli BL21 (DE3) after identified by PCR and doubly digested. The inclusion bodies of fusion protein were extracted and purified by washing, dissolved in 6 mol/L guanidine buffer, and refolded by rapid dilution technique. The refolding protein activity was tested by ELISA and flow cytometry. Results Extraceilular domain of the huFcγRⅡa gene was successfully cloned into pET-28a. The results of SDS-PAGE showed that the molecular mass (Mr) of the expressed protein was 24.8 × 103, and the expression rate was 30%. The purity of recom-binant shuR Ⅱ was up to 90% after washing. ELISA showed that the recombinant shuR Ⅱ was able to bind human IgG in a dose dependent manner, shuRⅡ could competitively inhibit the binding of human IgG to huFcγRⅡa expressed on the surface of COS-7 cells by flow cytometry. Conclusion The results demon-strate that it is possible to obtain large quantities of recombinant shuR Ⅱ which has comparable binding prep-erties to those of the whole membrane bound huFcγR Ⅱ a.

Block polystyrene-b-poly(acrylic acid) (PS-b-PAA) copolymers were obtained by means of atom transfer radical polymerization (ATRP), and then were covalently grafted to y-(2,3-glycidoxypropyl)trimethoxysilane (GPS) modified Si surfaces to obtain the solvent surfaces. GPC, FTIR, H-1 NMR and AFM were used to characterize the samples and surfaces. The surface responsive behaviors were investigated by water contact angle (CA) measurements. It shows that the wetting properties of the surfaces were dependent on the compositions of grafted copolymer and the properties of the solvents. The surface modified with the compolymer, which has lower PS/PAA ratio, is more hydrophilic under the same treatment. Hydrophilic surfaces were obtained after the treatments of ethanol and basic aqueous solution, while toluene and acidic aqueous solution treatments would result in the surface hydrophobicity. Moreover, the switching behaviors of the surface were also proved to be very stable with the external acid-base stimuli.

This article presents a brief overview of label-free optical biosensor-based cell assay technologies. Theoretical analysis suggests that a resonant waveguide grating (RWG) biosensor detects ligand-induced dynamic mass redistribution (DMR) within the bottom portion of an adherent cell layer. Pharmacological studies suggest that the DMR signal can serve as a novel physiological readout for monitoring receptor activation, and examining ligand pharmacology. Chemical biology studies support the hypothesis that the DMR signal is an integrated response that consists of contributions from many cellular events induced by the ligand, thus providing alternative means to study cell systems biology. Orthogonal confirmations using conventional cell biology approaches have led to identifying specific cellular event(s) that dominate the DMR signals observed for three classes of receptors: epidermal growth factor receptor, Gq-coupled receptors and Gs-coupled receptors.

Receptors for the Fc region (FcγRs) of IgG play a crucial role in the immune system and host protection against infection. In the present study, we describe the cloning, sequencing and characterization of porcine FcγRII. By screening a translated EST database with the protein sequence of the human FcγRII (CD32) we identified a putative porcine homologue. Using rapid amplification of cDNA ends (RACE), we isolated the full-length cDNA encoding porcine FcγRII from peripheral blood leucocyte RNA. The porcine FcγRII cDNA was 1488bp long, encoding a 297 amino acid trans-membrane glycoprotein composed of two immunoglobulin-like extracelluar domains, a trans-membrane region and a cytoplasmic tail with an immunoreceptor tyrosine-based inhibitory motif (ITIM). The predicted amino acid sequence was found to be 67% and 52% identitical with human and mouse FcγRIIB. RT-PCR indicated porcine FcγRII transcripts expressed in liver, alveolar, mesenteric lymph node and PBLs. COS-7 cells transfected with the pig FcγRII cDNA were able to bind chicken erythrocytes sensitized with porcine IgG. Identification of porcine FcγRII will aid in the understanding IgG-FcγR interactions, and may help in developing new immunization protocols.

To identify the linear epitope for Fc‐binding on the bovine IgG2 Fc receptor (boFcγ2R), peptides derived from the membrane‐distal extracellular domain (EC1) of boFcγ2R corresponding to the homologous region of human FcαRI were synthesized. Binding of bovine IgG2 to the different peptides was tested by Dot‐blot assay, and the peptide showing maximal binding was further modified by truncation and mutation. The minimum effective peptide 82FIGV85 located in the putative F–G loop of the EC1 domain was found to bind bovine IgG2 specifically and inhibit the binding of bovine IgG2 to the receptor. The Phe82, Ile83 and Val85 residues within the linear epitope were shown to be critical for IgG2‐binding. Such functional epitope peptide should be very useful for understanding the IgG‐Fcγ interaction and development of FcR‐targeting drugs.

The VP3 gene about 893bp was obtained from the total RNA of the chicken IBDV by RT-PCR, then the VP3 gene was subcloned into the downstream of T7 promoter of the vector pET-28a to construct expression vector of pETVP3. The recombinant plasmid was transformed into in E.coli BL21(DE3) and induced with IPTG. SDS-PAGE and Western blotting analyses showed that the chicken IBDV VP3 protein of 35.5 and 33 kDa were efficiently expressed and mainly existed as inclusion bodies,with specific reactivity to anti-IBDV antibodies.

Infectious bursal disease virus (IBDV) is the causative agent of infectious bursal disease, which is one of the most important and widespread infectious diseases in commercial chickens. Conformational epitopes have been reported in the highly variable region of the VP2 protein of IBDV. In the present study, a random heptapeptide library was screened by using monoclonal antibodies (mAbs), YNW17 and YNW29, directed to the VP2 of IBDV and two peptide motifs, D-X-P-R and A-R-G, were identified. The motifs are present on the N and C terminal sequences of the highly variable region of VP2. Synthetic overlapping peptides covering the motifs on VP2 were analyzed by Dot- ELISA with the mAbs and two epitopes 197CDSSDRPRVYTIT209 and 329ARGSLAVTI337 identified. The above epitopes were also recognized by chicken anti-IBDV sera and shown to inhibit the binding of their mAbs to recombinant VP2. Both mAbs and sera from mice immunized with the conjugated epitope-peptides were able to neutralize serotype I IBDV. These results indicated that the epitopes are two neutralizing linear B-cell epitopes and would be useful for the development of peptide-based IBD vaccines.

Replisome DNA primases are responsible for the synthesis of short RNA primers required for the initiation of repetitive Okazaki fragment synthesis on the lagging strand during DNA replication. In bacteriophage T4, the primase (gp61) interacts with the helicase (gp41) to form the primosome complex, an interaction that greatly stimulates the priming activity of gp61. Because gp41 is hexameric, a question arises as to whether gp61 also forms a hexameric structure during replication. Several results from this study support such a structure. Titration of the primase/single-stranded DNA binding followed by fluorescence anisotropy implicated a 6:1 stoichiometry. The observed rate constant, k(cat), for priming was found to increase with the primase concentration, implicating an oligomeric form of the primase as the major functional species. The generation of hetero-oligomeric populations of the hexameric primase by controlled mixing of wild type and an inactive mutant primase confirmed the oligomeric nature of the most active primase form. Mutant primases defective in either the N- or C-terminal domains and catalytically inactive could be mixed to create oligomeric primases with restored catalytic activity suggesting an active site shared between subunits. Collectively, these results provide strong evidence for the functional oligomerization of gp61. The potential roles of gp61 oligomerization during lagging strand synthesis are discussed.